653 organ specific oncolytic virus incorporated antigen libraries (OSOVIAL) for the immunotherapy of cancer

653 Organ Specific Oncolytic Virus Incorporated Antigen Libraries (OSOVIAL) for the Immunotherapy of Cancer Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene[.]

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We also observed a signi cant increase in the number of HIV-Gag

speci c T-cells that secrete IFNγ and IL-2 after ex vivo stimulation

with HIV/Gag derived peptides (1600 vs 800 for IFNγ, and 300

vs 120 for IL-2 SFC/106 splenocytes) In addition, an in vivo CTL

assay con rmed that vaccination with the EAT-2 overexpressing Ad

resulted in a signi cantly improved in vivo cytolytic activity of HIV/

Gag speci c CD8+ T cells generated after Ad-EAT2 and Ad-HIV/

Gag co-injection (68% vs 39% speci c killing) Since both mice

and humans express highly homologous EAT2 adaptors, our results

suggest that human vaccination strategies that speci cally facilitate

SLAM receptor signaling may provide a more effective vaccine

against HIV speci cally, as well as numerous other vaccine targets

in general

Cancer – Immunotherapy II

652 A Powerful Retroviral Vector for TCR

Gene Therapy: Silencing of Endogenous TCR

Improved Expression and Anti-Tumor Reactivity

of Transduced Tumor-Speci c TCRs in Human

Lymphocytes

Sachiko Okamoto,1 Hiroaki Ikeda,2 Hiroshi Fujiwara,3 Masaki

Yasukawa,3 Hiroshi Shiku,2 Junichi Mineno.1

1 Center for Cell and Gene Therapy, Takara Bio Inc., Otsu, Shiga,

Japan; 2 Department of Immuno-Gene Therapy, Mie University

Graduate School of Medicine, Tsu, Mie, Japan; 3 Dept of

Bioregulatory Medicine, Ehime University Graduate School of

Medicine, Toon, Ehime, Japan.

Adoptive T cell therapy with genetically engineered lymphocytes

to express tumor antigen speci c T-cell receptor (TCR) has been

shown as an attractive strategy to treat cancer patients However,

the limited ef cacy of TCR gene therapy has been reported to be

associated with the inef cient surface expression of transduced TCRs

in T lymphocytes Endogenous TCR competes with introduced TCR

for CD3 molecules and reduce the cell surface expression of the

introduced TCR In addition, the introduced TCR α and β chains have

been reported to mispair with endogenous TCR subunits, resulting in

insuf cient formation of heterodimers of therapeutic TCR Moreover,

misparing of endogenous and transduced TCR subunits may cause

the generation of T cells with unexpected speci cities, including a

risk of autoreactivity

In this study, we developed novel “siTCR” retroviral vectors

encoding both siRNA constructs that specifically knockdown

endogenous TCRs and codon-optimized, siRNA-resistant TCR αβ

chains speci c for human tumor antigens Human lymphocytes

transduced with these vectors exhibited high expression of the

introduced tumor-speci c TCR on the cell surface accompanied with

reduced endogenous TCR at low copy numbers of the integrated

vector, resulting in enhanced cytotoxic activity against

antigen-expressing tumor cells As the target of this novel strategy is

endogenous TCR, siTCR vectors may be a powerful tool for any TCR

gene therapy without any dependency of TCR variation To apply this

novel siTCR vector strategy to any TCR variations, we also developed

universal siTCR retroviral vector cassette, which made possible to

construct any siTCR retroviral vector with suitable TCRs

This new approach satisfies the following requirements:

enhancement of the ectopic TCR heterodimer expression on the

cell surface and enhanced biological function at low proviral copy

number (which may reduce the risk of mutagenesis); reduction of

the endogenous TCR expression, which may effectively reduce

TCR mispairing and decrease the risk of inducing self-reactive TCR

αβ heterodimers; a universal method that does not depend on TCR

variation Therefore, our novel TCR gene therapy may open the new

gate for effective immunotherapy in cancer patients

653 Organ Speci c Oncolytic Virus Incorporated Antigen Libraries (OSOVIAL) for the Immunotherapy of Cancer

Jose Pulido,1,2 Timothy Kottke,2 Jill Thompson,2 Peter Selby,3 Alan Melcher,3 Richard Vile.2,3

1 Ophthalmology and Ocular Oncology, Mayo Clinic, Rochester, MN; 2 Molecular Medicine, Mayo Clinic, Rochester, MN; 3 Cancer Research UK Clinical Centre, St James’ University Hospital, Leeds, United Kingdom.

AM and RV are joint senior authors We have previously developed an immunotherapy for cancer in which normal cells are intentionally killed in the presence of the potent adjuvant hsp70

to induce autoimmune T cell reactivity In turn, this autoimmune reactivity is effective against tumors expressing normal tissue

speci c antigens of the same histological type (Cancer Res 2009

69:7767) Using viral vectors to deliver cytotoxic/hsp70 genes to the appropriate normal tissues, we have shown that this approach can effectively treat established melanoma, prostate cancer and pancreatic cancers in murine models However, there are concerns about inducing intentional destruction of normal tissues to treat malignancy in cases where such damage could have pathological consequences (such as with the pancreas) We have also shown that oncolytic viruses, such as VSV, are powerful immunoadjuvants for induction of T cell responses against tumor associated antigens

(TAA), if a) the virus leads to release of TAA in the lymph node (Nat

Med 2008 14:37; Clin Cancer Res 2009 15:4374) and b) when the virus itself encodes the TAA (Cancer Res 2007 15:2840) Therefore,

we hypothesized that it would be possible to combine induction of autoimmune reactivity against normal tissue speci c antigens with highly immunogenic, viral adjuvant-induced antigen presentation, for cancer immunotherapy To achieve this, we cloned cDNA libraries from a variety of sources, including normal tissues, into oncolytic viruses such as VSV We reasoned that the viral-expressed cDNA library would encompass epitopes from most normal antigens which might serve as immunogens for cancer rejection, thereby dispensing with the need for direct killing of the normal tissue In addition, the immunogenicity of the VSV would serve the adjuvant properties

of hsp70 We have now shown that intravenous injection of VSV encoding a cDNA library from normal prostate leads to regression of murine prostate tumors in immune competent mice Regressions were immune mediated, as opposed to induced by viral oncolysis Transient in ammation was observed in the normal prostate but no evidence of long term autoimmunity was apparent Mice in which tumors were rejected developed potent Th17 responses against prostate tumor cells,

as well as normal prostate tissues Moreover, tumors which initially

regressed but were not cured, recurred aggressively, with a markedly different histological appearance, and antigenic profile, to the initially implanted tumor cells, suggesting that OSOVIAL-mediated vaccination imposes a stringent immune selection upon tumors This approach dispenses with the need to induce direct damage to normal

tissues in situ, allows for wide-ranging in vivo immune selection for

antigens which are likely to be effective targets for both autoimmune and anti tumor responses and uses oncolytic viruses as novel immune adjuvants for the immunotherapy of cancer

654 Editing Human Lymphocyte Speci city for Safe and Effective Adoptive Immunotherapy of Leukemia

Elena Provasi,1 Pietro Genovese,2 Zulma Magnani,1 Angelo Lombardo,2 Andreas Reik,3 Liu Pei-Qi,3 Oscar Muniz Pello,2

Jurgen Kuball,4 Attilio Bondanza,1 Giulia Casorati,1 Philip D

Gregory,3 Claudio Bordignon,1 Michael C Holmes,3 Philip D

Greenberg,4 Luigi Naldini,2 Chiara Bonini.1

1 San Raffaele Scienti c Institute, Milan, Italy; 2 HSR-TIGET, Milan, Italy; 3 Sangamo BioSciences, Richmond, CA; 4 Fred Hutchinson Cancer Research Center, Seattle.

T cell receptor (TCR) gene-transfer is an attractive strategy for the adoptive immunotherapy of tumors However, the full potential of this approach is limited by a number of technical hurdles including inef cient gene transfer, unstable transgene expression, exhaustion of gene-modi ed cells and, most importantly, the co-expression in the same cell of the endogenous and tumor-speci c TCR The latter results not only in reduction of expression of the introduced tumor-speci c TCR, but also can result in acquisition of autoreactive speci cities due to mispairing between endogenous and exogenous TCR chains

To address these limitations, we designed a novel strategy based

on zinc  nger nucleases (ZFNs) and lentiviral vectors that allows for the  rst time editing of T cell speci city at the DNA level, by combining the disruption of the endogenous TCR β chain gene with the transfer of a tumor-speci c TCR We  rst stimulated PBL with anti-CD3 and anti-CD28 antibody-conjugated beads, and cultured the cells with low doses of IL-7/IL-15, to target and preserve early differentiated T cells To eliminate expression of the endogenous TCR, activated cells were treated with integrase defective lentiviral vectors (IDLV) carrying a set of ZFNs speci c for the constant regions of the TCR β chain Abrogation of surface expression of the TCR/CD3 complex was observed in up to 7% of the treated cells

The majority (>80%) of CD3neg lymphocytes expressed CD62L, CD127, CD27 and CD28, a phenotype most consistent with central memory T cells, and could be expanded in culture with IL7 and IL15

as unmodi ed T cells To re-direct tumor speci c T cell activity, we selected a codon-optimized, cysteine-modi ed TCR speci c for the Wilm’s Tumor Antigen 1 (WT1), which is involved in oncogenic transformation in several tumors To promote balanced expression of the introduced α and β TCR chains, we generated lentiviral vectors (LV) encoding both chains under a single PGK bidirectional promoter (PGK-WT1 LV) Sorted CD3neg cells were ef ciently transduced (>45%) with PGK-WT1 LV, and expressed stable and high levels

of tumor-speci c TCR, indicating that CD3neg cells are permissive

to further genetic manipulation TCR-edited cells were enriched

to 90% purity by polyclonal stimulation, indicating that surface expression of the transferred TCR/CD3 complex was functional and suf cient to promote cell expansion TCR-edited cells killed targets pulsed with lower peptide concentrations than unedited T cells, modi ed by conventional TCR gene transfer, and lysed fresh WT1+/HLA-A2+ primary acute myeloid leukemia blasts These data demonstrate that genetic re-programming of T cell speci city in early differentiated lymphocytes is feasible and functional and represents

an attractive approach to improve the therapeutic outcome of cancer immunotherapy *equal contribution

655 Constitutive 4-1BBL Expression Dramatically Increases Proliferation, Survival and

In Vivo Persistence of CD19-Targeted T Cells, Promoting Complete Tumor Elimination

Maud Condomines, Jason Plotkin, John C Markley, Gertrude Gunset, Michel Sadelain

Center for Cell Engineering, Memorial Sloan-Kettering Cancer Center, New York, NY.

Human T cells engineered to express a chimeric antigen receptor (CAR) composed of an antibody-derived scFv fragment linked to the CD3 zeta chain, can recognize and kill tumor cells expressing the targeted membrane antigen However, T cell activating signals have

to be enhanced by a second stimulatory signal in order for T cells to avoid anergy, to be fully activated, proliferate and secrete cytokines CD80 and CD86 are the prototypical costimulatory molecules, which bind the CD28 receptor constitutively expressed at the T cell plasma membrane Activation of 4-1BB, a member of the TNFR family expressed by activated T cells, has been shown to ef ciently enhance CD8 T-cell proliferation and mediate increased tumor rejection in animal models We previously showed that the retroviral transfer

of an anti-PSMA CAR (Pz1) along with CD80 and 4-1BB ligand (4-1BBL) into human T cells induced auto-costimulation enabling high in vivo T cell expansion and tumor rejection in a prostate tumor model In this study, we investigated this concept in T cells harboring either a  rst generation anti-CD19 CAR (19z1) or a second generation CAR (1928z), which additionally comprises the CD28 cytoplasmic domain We compared the proliferation and anti-tumor abilities of 19z1+, 19z1-4-1BBL+, 19z1-CD80+4-1BBL+, 1928z+, 1928z-4-1BBL+ transduced T cells in an aggressive pro-B cell leukemia model (NALM-6)

After 3 weekly in vitro stimulations on mouse fibroblasts expressing human CD19, 19z1+, 19z1-4-BBL+, 19z1-CD80+4-1BBL+, 1928z+ and 1928z-4-1BBL+ T cells exerted a 2-fold, 5-fold, 21-fold, 17-fold and 200-fold expansion rate, respectively All T cell groups killed four CD19 expressing cell lines, including NALM-6, with similar ef ciencies The proportions of central memory CD8 T cells (CD45RA- and CCR7+) observed after one stimulation in the

3 groups expressing 4-1BBL were 40-60% whereas those evaluated

in the 19z1 and 1928z groups were only 20-25% One week after IV injection of 1e6 CAR+ T cells into 4-day NALM-6 bearing NOD/ SCID/IL2Rγ null mice, the proportion of human CD8 and CD4

T cells which had accumulated in the bone marrow measured in mice infused with 1928z-4-1BBL+ T cells was 100-fold and 10-fold higher than in mice infused with 19z1+ and 1928z+ T cells, respectively The proportions of CD8 and CD4 T cells in the spleen followed those observed in the bone marrow, including a substantial proportion of central memory T cells One month after T cell injection, 1928z-4-1BBL+ CD8 T cells expanded at least 300 times In vivo bioluminescence imaging showed complete tumor regression in the 1928z-4-1BBL+ T cell-treated group within 15 days of T cell infusion, while a partial regression was observed in the other groups Long-term survival studies are on-going

Thus, constitutive 4-1BBL expression drives high proliferation of anti-tumor CD8 T cells, which accumulate in the spleen and bone marrow of immuno-compromised, tumor-bearing mice, promoting central memory T cell persistence and tumor eradication

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